Interconnected networks of cells in the brain called neurons underlie all cognitive functions. Key advances in our understanding of brain function during the last several decades have resulted from technologies that permit monitoring of electrical activity in neurons. This technique, broadly referred to as electrophysiology, permits the study of circuits of connected neurons responsible for sensation, movement, thought, learning, and memory. These techniques have also revealed how abnormal electrical signaling between neurons can lead to dysfunction as occurs in disorders such as autism or Alzheimer's disease as well as due to damage from a stroke or traumatic injury. The most sensitive form of electrophysiological recording monitors very small electrical currents in single cells with glass pipettes placed inside the neuron. These 'intracellular' patch-clamp recordings are a powerful tool for exploring how neurons work-and don't work. Despite these great advances, current intracellular recording technology has significant limitations: puncturing the cell damages it, leading to short recordings and abnormal biophysical and biochemical properties; skilled scientists are required, reducing the number of labs that can use this technique; and simultaneous recording from more than one or two neurons is rarely possible, making it challenging to study neuronal communication. Stealth Biosciences was established to overcome fundamental limitations of existing intracellular techniques. Our group invented a new technology which we call Stealth or biomimetic electrodes. These electrodes are able to fuse into the cellular membrane, providing a minimally damaging, electrically tight junction with the cell. Our initial measurements have demonstrated high-quality, long-term intracellular recordings that rival that of traditional patch-clamps. Biomimetic probes are based on standard silicon microfabrication processing, enabling large arrays of electrodes on inexpensive chips. Using support from a Phase I SBIR grant, we will refine the device and perform feasibility studies of this game changing, inexpensive, and easy-to-use intracellular recording platform. Our long-term goals are to develop this technology for wide commercial distribution among researchers to advance basic discoveries, accelerate drug development, and improve the health and well-being of those suffering from disorders of the brain. To achieve this ambitious program we outline two Phase I Specific Aims: Aim 1: Optimize Biomimetic Electrode Performance and Production In this Aim, we will assess the performance of different geometric and architectural designs for biomimetic electrodes. Designs will be evaluated for electronic characteristics as well as for electrophysiological performance with cultured neurons. The fabrication process will be streamlined and structured with the goal of eventual large-scale fabrication. Specific milestone goals include electrical performance of < 2mV noise, better than 0.1 ms time resolution, and <200MW input impedance. Timing: Q2 and Q3. Aim 2: Functional Characterization of Biomimetic Probes with Cells in Culture The second Aim will characterize the biomimetic device performance for recording from rat hippocampal neurons. This stringent test of intracellular recording capabilities will allow direct comparison to the gold- standard pipette-based patch-clamps. Cell recordings from the different probe designs in Aim 1 will be used to optimize fabrication techniques and probe design. Long-term recordings extending for days and possibly weeks will be used to demonstrate lifetime and temporal capabilities of the probes far exceeding what is possible with conventional patch-clamps. Timing: Q3 and Q4. We have brought together a strong team with expertise in electrophysiology, micro/nanofabrication, cell-to-cell communication, and business to support this technology development at Stealth Biosciences. At the end of this program, we will have an experimentally vetted system for 'turnkey' intracellular recordings. These will provide simple cell-preparation, >16 individually addressable electrodes per chip, high-quality recordings, and compatibility with existing electrophysiological software and recording hardware. We believe these devices will find broad interest within the neuroscience community, both as a basic research tool, and for advanced applications in drug discovery and personalized medicine.